Abstract
The dominant paradigm of protein engineering is structure-based site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including beta-galactosidase, alkaline phosphatase, and beta-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be "reprogrammed" for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.
Highlights
Rational protein design is generally synonymous with structure based site-directed mutagenesis [10]
Reporter proteins are usually selected as starting points for sensor design because their structurehave been solved and because their activities are amenable to high throughput screening
Protease Activation of p53 In Vivo Activation—We first engineered p53 variants that were activated by HIV protease or the B. anthracis Lethal Factor
Summary
Materials—Expression vectors pET20bϩ, pET28aϩ, and pCDF Duet were from Novagen (Madison, WI). The lacZ gene was excised from expression vector p1 ϩ IQ [30] using BamHI; the remaining DNA was purified, self-ligated, and used to transform E. coli strain Inv␣FЈ. The HIV protease gene in p1ϩIQ was PCR amplified, subcloned into pET28aϩ using NdeI and Hind III, and sequenced to confirm its wild-type identity. Protein Purification—E. coli BL21 (DE3) cells containing the plasmid pLysS were transformed with constructs that expressed the wild-type or engineered p53 genes fused to N-terminal six-histidine tags. The wild-type and engineered p53 proteins were expressed, either alone or co-expressed with HIV protease (from p1ϩIQ [30]) or LF protease (from LF-pCDF), and purified by immobilized metal affinity chromatography (IMAC) as described above. The in vitro protease assays were performed using purified p53 (p53/p6, p53/LF10, p53⌬30, or wild-type) and protease (HIV protease [31] or Lethal Factor) proteins. The filters were washed four times more for 7.5 min each, and the quantity of probe bound to each filter was measured using a BAS-1000 Bio-imaging Analyzer System (Fujifilm Medical Systems USA, Stamford, CT)
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